The potential value of cuprotosis in myocardial immune infiltration that occurs in pediatric congenital heart disease in response to surgery with cardiopulmonary bypass

Abstract Background Cardiopulmonary bypass may cause malfunction in the myocardium. Cuproptosis is a novel cell death aggregating mitochondrial proteins. However, the research on cardiopulmonary bypass‐caused heart tissue injury in immune infiltration and cuproptosis is limited. Method Immune infiltration, enrichment analysis, protein−protein interaction network, and medication prediction are applied to reanalysis differentially expressed genes and cuproptosis‐related genes in gene expression omnibus data set GSE132176. Results Seven cuproptosis related genes (PDHA1, LIPT1, LIAS, DLST, DLD, DLAT, and DBT) and dendritic cells and Th1 cells are involved in heart tissue injury in response to surgery with cardiopulmonary bypass. Conclusions Immune infiltration and cuproptosis are potential mechanisms by which cardiopulmonary bypass surgery may cause damage to heart tissue, which may be a new therapeutic target.

Cuproptosis is a new kind of programmed cell death in which an accumulation of intracellular copper contributes to the aggregation of mitochondrial proteins and destabilization of Copper cluster proteins, most likely resulting in a specialized form of cell death. 6 Previous studied shows that the myocardial copper concentrations increased during the aortic crossclamping period in the heart surgery. 7 The change of myocardial copper ion concentration is closely related to the impact of cardiac surgery on myocardial cells. Therefore, we assume that copper death participates in and affects the process of myocardial injury during cardiopulmonary bypass. The study of copper toxicity and cuproptosis-related genes (CRGs) is still in its infancy and there is an urgent need for more research in this field. Microarrays have been utilized in recent years to examine the expression of various organ differential genes (DEGs) before and after therapy with CPB. Liang and coworkers established a network regulatory relationship between Long noncoding RNA (LncRNA) and transcription factors in the injured rat brain after surgery with CPB 8 and Ghorbel et al. concentrated on differential gene expression in the renal medulla using a well-established model of acute kidney damage after surgery with CPB. 9 We reanalyzed the data set based on Raggi et al.'s sequencing data to analyze tetralogy of Fallot (TOF) and atrial septal defect (ASD) specific myocardial transcriptional signatures in the gene expression omnibus (GEO) data set and bioinformatics permits us to explore how these genes function in the pathophysiology of heart tissue injury after CPB. 10 2 | METHODS AND MATERIALS

| Data set collection and normalization
This microarray GSE132176 (https://www.ncbi.nlm.nih. gov/GEO/platform/GSE132176) is comprised of 10 right atrial samples with ASD and TOF for expression study before and after surgery with CPB and all samples were incleded in our study. Annotation of raw data is used to map probes to their associated gene symbols. When numerous probe sets are associated with a single gene, the expression levels of each probe within the multiple probe sets are averaged and ascribed to a single gene. The data were then Log2 transformed and Z-score normalized. Networkanalysts were used to construct boxplots, principal component analysis (PCA) paragraph, count sum, density plot, and mean squared deviation (MSD) plots. 11

| Screening for DEGs and CRGs
DEGs were identified using the limma package in R. The ggplot2 and pheatmap packages were used to constructed volcano map and heatmap of DEGs, respectively. DEGs with p < .05 and |log 2 FC| >1 were considered as being significantly different.
The unpaired T-test is used to examine the fragments per kilobase of transcript per million fragments mapped (FPKM) values of genes related with cuproptosis-induced cell death.
Enrichr and metascape are web-based tools for performing intuitive enrichment analysis with many visualization kinds and gene collection functionalities. 14,15 Gene set enrichment analysis (GSEA) version 4.1.0 software was used to analyze genes function from the GSEA website MsigDB database (http://software. broadinstitute.org/gsea/msigdb). The default weighted enrichment method was applied for enrichment analysis. The random combination was set for 1000 times. GO and KEGG pathway AND reactome pathway enrichment analysis were performed with GSEA and FDR < 0.25, NOM p < .05, and |NES| >1 were considered significant enrichment.

| Protein−protein interaction (PPI) network construction and hub gene identification
Online database STRING (available online: http://stringdb.org) 16 was employed to develop DEGs-encoded proteins and PPI network. Cytoscape software 17 was used to establish a network of protein interaction relationships and to assess the protein interaction relationships of the putative DEGs. On Cytoscape, Molecular Complex Detection (MCODE) 18 was used to identify functional clusters of genes in the PPI network with the parameters degree cutoff = 2, node score cutoff = 0.2, k-core = 2, and maximum depth = 100.
The proteins matching to the center nodes may be core proteins and candidate genes with crucial physiological regulatory roles.

| Immune infiltration analysis and correlation analysis with cuproptosis
Using the "GSVA" and "ssGSEA" R packages, we can analyze the infiltration score of 16 distinct immune cells, the activity of 13 distinct immune-related activities and the activity of 40 distinct immunological-related checkpoints. In addition, correlation analyses between cuproptosis genes, immune cells, and immune-related activities are generated and shown using the "corplot" and "heatmap" R packages.

| Statistical analysis
As mentioned in the figure legends, the data are presented as means with standard deviations and the student's t-test is used to examine significant differences.
In addition, the Mann−Whitney test with p values adjusted by Benjamini−Hochberg is used to compare the ssGSEA scores of immune cells or pathways between the high risk and low risk groups. Moreover, the statistical significance level is set at p < .05. GraphPad Prism (Version 8.0.2), ggpubr, and reshape2 are also utilized to analyze the data and provide the appropriate graphics.

| Standardization of data and identification of DEGs
The primary goal of normalization is to remove technical and systematic data variability so that comparisons may be made across samples.
Probe counts ( Figure 1D) were first summed for heart tissues before and after CPB respectively. Afterwords during normalizing the microarray data, the gene expressions of each sample are shown in the boxplot ( Figure 1A,B) and the black lines in the boxes are virtually on the same straight line, verifying the raw data's correctness.
PCA ( Figure 1C) paragraph which assess the biological variation across samples after normalization shows two group are distinguished apparently. In addition, the lines of two group in density plot ( Figure 1E) which facilites comparison of signals between arrays and identification of arrays with deviating distributions depits similar to each other. MSD plots ( Figure 1F) which created for individual trajectories fit it to a straight line which indicated further the potiential value of analysis.
Significant DEGs were identified among post-CPB heart tissues compared with before-CPB ones. A total of 2937 DEGs were screened out, including 1504 upregulated DEGs and 1433 downregulated DEGs. These data were used to build the volcano plot of DEGs ( Figure 2A) and the heatmap of the top 50 genes ( Figure 2B).

| Enrichment and PPI network analysis of the DEGs
The enrichment analysis revealed that there were markedly enriched in response to lipopolysaccharide (p < .01), response to molecule of bacterial origin (p < .01), cell chemotaxis (p < .01), and leukocyte chemotaxis (p < .01) in BP ( Figure 3A) and there were also significant enriched in DNA-binding transcription activator activity (p < .01), RNA polymerase II-specific (p < .01), and DNA-binding transcription factor activity (p < .01) in MF ( Figure 3B). In the meanwhile, there were markedly enriched in collagen-containing extracellular matrix (p < .01) in CC enrichment ( Figure 3C). What's more there were markedly enriched in TNF signaling pathway (p < .01), pertussis (p < .01), and complement and coagulation cascades (p < .01) in KEGG pathway ( Figure 3D).
Afterwords, A PPI network of DEGs that included 406 nodes and 1097 interactions was constructed to identify gene interactions ( Figure 5A). Module analysis identified significant clustering modules in the PPI network ( Figure 5B). The three highest-scoring clustering modules were obtained and each hub gene was found in ≥1 module. Thus, the eight clustering modules may represent key biological roles of the PPI network.
Enrichr tool is used to perform enrichment analysis and predict the drugs with genes involved in copperrelated cell deaths and immune infiltration.
The majority of the enriched results in MF are for the following functions: acyltransferase activity, transferringgroups other than amino-acyl groups (GO: 0016747) (p < .05), oxidoreductase activity, acting on a DONOR group, NAD (P) as acceptor (GO: 0016668) (p < .05), and oxidoreductase activity, acting on the aldehyde or oxo group of donors, disulfide as acceptor (GO: 0016624) (p < .05) ( Figure 6C).
In the meanwhile, when analyzing genes involved in copper-related cell death, the KEGG pathway enrichment analysis found that the citrate cycle (TCA cycle) (p < .05), pyruvate metabolism, and glycolysis/ gluconeogenesis (p < .05) are most heavily involved ( Figure 6E).

| Immune infiltration analysis
More specifically, we used ssGSEA and GSVA to quantify the enrichment scores of various immune cell subpopulations, associated functions, or pathways and the links between risk score. Figure 7A shows the heatmap of immune cells and functions. Figure 7B revealed correlation heatmap of the 15 types of immune cells. CD8+ T cell has a high positve correlation with TIL. DCs has a high positve correlation with Th1 cell macrophages has a high positve correlation with Til. Neutrophils has a high positve correlation with Tregs. Tfh has a high positve correlation with Th1 cell. Figure 7C revealed correlation heatmap of the 13 types of immune funtions. CCR has a high positve correlation with inflammation-promoting and MHC class-I. Check-point has a high positve correlation with inflammation-promoting. Cytolytic-activity has a high positve correlation with HLA. HLA has a high positve correlation with T cell coinhabitation. Inflammationpromoting has a high positve correlation with parainflammation. Inflammation-promoting has a high positve correlation with type I IFN response.

| Enrichment analysis, medication prediction, correlation between CRGs and immune infiliatration
LIPT, LIAS, DLST, DLD, DLAT, DBT, and PDHA1 are all negatively associated with immune cells and functions associated with cuproptosis and the details are as follows ( Figure 9A). LIPT1, LIAS, DLST, DLD, and DLAT are negatively correlated with cuprotosis-related genes and immunological infiltration of aDCs and the DCs had an inverse relationship to LIPT1. In addition the pDCs have an inverse relationship to the DBT and DBT and T cell costimulation are in an inverse relationship. What's more, DBT also had a negative connection with proinflammatory factors and it is shown that CCR is inversely related to LIPT1, DBT. Next, there is an inverse relationship between MHC class I and LIPT1, DLD, DLAT, and DBT and between parainflammation and DBT. However, There is also an inverse relationship between CHECKPOINT and PDHA1 and between type I IFN response and DBT.

| DISCUSSION
In our study, we explore the likely function of immune infiltration and genes related with cuproptosis in heart tissue injury following CPB for the first time. Despite the substantial risk of postoperative inflammation and organ injury, CPB are becoming more and more popular in treating congenital heart defects. [19][20][21][22][23][24] Clinically unfavorable outcomes of cardiopulmonary bypass might range from minor symptoms to life-threatening complications and heart failure and low cardiac output syndrome is the most severe consequence. 25 Conventional myocardial protection during surgical procedures focuses on reducing the inflammatory response 26 which is consistent with our enrichment analysis results. In addition to evaluating the significance of inflammation and the immune system, we perform an immune infiltration, the results of which reveal the significance of dendritic cells, Th1 cells, Apc CO inhibition, CCRinflammation promotion, MHC class I, and type II IFN responses. In the immune cell infiltration, previous research has revealed that after an acute myocardial infarction, DC are present in cardiac tissue and are related with significantly elevated HLA-DR levels. 27 In the meanwhile, T-helper cell differentiation and maturity are promoted in patients with heart failure, but B cell toxic cells are diminished. 28 This indicated that the combination of DCs and T cells may induce an immunological response that exacerbates cardiac injury. As to immune function infiltration, interferons with type II response are useful for treating heart failure in clinical trials. What's more, on the basis of the direct effects of IFN on cardiac fibroblasts, Hellkvist et al. discovered that IFN caused the synthesis of hyaluronic acid and stimulated the development of fusion cultures including cardiac fibroblasts obtained from rat hearts. 29 In addition to immunological and inflammatory factors, prior research also indicates that myocardial copper concentrations rose during the aortic crossclamping phase of heart surgery 30 and copper may be liberated from extracorporeally circulating blood during cardiopulmonary bypass. 31 Cuprotosis is a novel programmed cell death characterized by aberrant copper ion concentrations and abnormal citric acid cycle. 6 As to abnormal citric acid cycle, Aaron K. Olson's study demonstrated that triiodothyronine increases myocardial function and pyruvate entry into the citric acid cycle after reperfusion in a model of infant cardiopulmonary bypass. 32,33 Advancements in transition metal signalings have facilitated the translation of basic studies in copper chemistry and biology into therapeutic drugs and diagnostics by establishing novel interconnections. During congenital heart disease repair surgery, ferroptosis, the new metal signalings, is connected with mitochondrial dysfunction in cardiac tissue 34 and in our study, cuproptosis, another metal signaling, shows a strong correlation with mitochondrial damage. The differential CRGs in our study such as LIPT1, LIAS, and DLD (related genes of key enzymes of lipoic acid pathway) and DLAT and PDHB (related genes of pyruvate dehydrogenase complex) can significantly alleviate the cytotoxicity mediated by copper ion carrier where LIAS is the key genes in cuproptosis procedure. 7 Maintaining proper pyruvate metabolism, 35 anaerobic glycolysis, and glycogen decomposition increasing 36 and glycolysis and ketooxidation rates lifting 37 may be the reasons how the mitochondria was injuried.
Next, the data set is utilized to analyze the genes implicated in cuproptosis through immune infiltration; DBT, DLAT, DLD, LIPT1, and PDHC are demonstrated to have a significant inverse relationship with their related immune cells. As to the indicated clinical medicine, the DSigDB database of pharmacological compounds forests latamoxef, atrazine, vitinoin, chlorzoxazone, and clomipramine as essential medications. Latamoxef are evidence to perform anti-inflammatory functions. 38 Chlorzoxazone has negative correlation with plasma TNF and IL-6 levels in patients with congestive heart failure. 39 Moreover, clomipramine-treated infected mice exhibited less cardiac inflammation, tissue necrosis, and fibrosis than untreated infected animals. 40 This research presents a novel route of myocardial protection during CPB, despite significant limitation due to the limited sample size of the data set contained (10 instances with TOF and ASD) and the difference between TOF and ASD may also affect the result. Meanwhile due to the lack of sequences on myocardial injury after CPB, it is hard to add the verifying set to eliminate the difference in population and gene characteristics. Although there is no confirmed clinical evidence to support the DsiDB-predicted medication, interfering with cuprotosis-related cell death may extend the lives of CPB-experienced individuals. Subsequent research are needed to examine single-cell sequencing data from a larger population to better comprehend this phenomena.

| CONCLUSIONS
Our research demonstrates that immune infiltration and cuproptosis are the potential mechanisms by which cardiopulmonary bypass surgery may injure heart tissue, which might be a unique medicinal development target.